PCR Polymerase Chain Reaction

Transcription

1 PCR Polymerase Chain Reaction Solubiosysteemit S Harjoitustyö Maria Sipilä

2 Contents: 1. Introduction 3 2. Gene cloning in bacteria 3 3. The other way to multiply genes: PCR Tools for PCR The principle of PCR The Melting Point Temperature Annealing State Temperature What time does it take? Problems with primers Allocated mutagenesis 7 4. Other techniques and applications for PCR 7 5. Sources 8

3 1. Introduction Gene multiplying is needed for researching for example diseases or producing some protein. There are two main ways: Gene cloning in bacteria and polymerase chain reaction. First I m going to discuss the basic of gene cloning in bacteria and then I ll go a bit deeper with PCR. 2. Gene cloning in bacteria The main idea is to transpose a gene in some vector into a bacterium and then let it divide until there s enough bacteria (and genes also). Then gene is isolated, or kept in plasmides to have genebanks. Just plasmides are mainly used as segmentation places. Bacteria have also solute DNA but plasmides are easier to handle (they are easy to get off from cells and the extra DNA is easy to add into them). Plasmides can also move to another cell. Picture 1. Gene s multiplying in cells. Violet is transposed gene. When you clone gene in bacteria, you will need: restriction enzymes (they cut the stripes from an exact place leaving an unpaired base strand, normally from fore to eight bases), ligase enzymes (they glue the unpaired strands with reversed bases), DNA-polymerase enzymes (they douplicate the amount of DNA) and reverse transcriptases (which make cdna from mrna because bacteria don t know how to remove introns which would lead to an inactive protein). There are several ways to get the wanted gene in to a cell: plasmid vectors, virus vectors, micro injection and using electricity or chemical agent to brake the cell membrane.

4 3. The other way to multiply genes: PCR PCR was invented by Kary Mullis (USA) in He and another scientist get the Nobel prize in ten years later. PCR is nowadays used and is almost invaluable tool for gene researchers. The superiority in PCR is its quickness and cheapness compared to other processes. It needs only little model-dna to produce a huge range of copies. Mutations in primers can make ligase anzymes attach to it and so the gene is ready to transpose to a cell. PCR, however, needs unleast some information of the gene s DNA order to make the primers. It s possible to use same sort of gene and guess the primers: this will sometimes work. Because PCR is so sensitive, it is also very susceptible to surroundings impurity. 3.1 Tools for PCR To get PCR work, you need: a small amount of model DNA, DNA polymerace enzymes, nucleotides and primers. There are two different kind of primers, one for both strands. Primers are often about 20 nucleotides long. 3.2 The principle of PCR First: Clean the sample from proteins and other dirts Second: Separate the strands by heating up to Third: 95 C (called the melting point =T m ) Primers attach to strands and build new ones (called annealing T a = C) Fourth: The strands douplicate at their favourite temperature (+72 C) Then the prosess stars all over again Picture 2. The principle of PCR

5 3.3 The Melting Point Temperature Denaturation is the progress when the two strands separate each other. The temperature of this is called the melting point temperature (T m ). It depends on the number of guanine and cytocine (the more, the higher temperature) and also on the length of primers (the longer, the higher). An average length for primer is 2 µm. T m can be calculated: T m = 81,5 C + 0,41*(%G + %C) 550/n where n is the number of nucleotides. Usually for primers to work, T m < 2 C. 3.4 Annealing State Temperature Annealing state temperature is the stage where primers fasten the strands and the building begings (from the 3 end). The temperature is conditional on the concentration of primers and the composition of nucleotides. This stage last normally only few seconds, but it is often programmed to 0,5-2 min long. 3.5 What time does it take? Denaturation: sec Annealing: sec Doupling: sec There can be made only cycles, otherwise there will be errors. At the end of program the temperature will be 72 C for 5 minutes to get every DNA strand ready.

6 3.6 Problems with primers If primers are negligently Picture 3. The hairpin structure designed, there may occur some unwanted structures. These can be prevented using some computer programs which calculate the possibility of those structures to form. One usual this kind of structure is the hairpin structure. This happens when the primers 3 end and 5 end pairs, or the other end pairs with the body. Another structure is primer dimers where the primers Picture 4. The primer dimer structure bind themselves and then form the rest of the structure. Commonality to both of these structures is that only if the 3 end is involved to the structure, only then will the primer be useless. There may also be nonspesific products because of false adhesion of primers. They are possible if only few bases are wrong or T a is too low.

7 3.7 Allocated mutagenesis Picture 5. Amino acid table As said before, if there is one or two false basepair on a strand, it doesn t prevent the primer to bind. This can be useful for reforming proteins: a certain base can be traced out or change a base to another to improve toleranse for cold or hot. A silent mutation is called an event, where the base order is changed, but the peptide order isn t. This is possible because there is several different triple codons for example for arginine, when changing one base from strand won t change the peptide. 4. Other techniques and applications for PCR PCR has also went forwards: there has been developed several of methods to improve the mechanics of PCR. To name a few, non-symmetric PCR produces one-stranded DNA for sequencing; inverced PCR copies some unknown piece of DNA-strand between 2 known ones. There has been also invented primers to recognize genetic diseases and so on

8 5. Sources: Ulmanen, Tenhunen ym. Geeni Biologia, WSOY, Porvoo Pictures: Picture 2: Ulmanen, Tenhunen ym. Geeni Biologia, WSOY, Porvoo 2000 Picture 3 & 4: Picture 5: CIRCLE.jpg